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MED19 and MED26 are Synergistic Functional Targets of the RE1 Silencing Transcription Factor in Epigenetic Silencing of Neuronal Gene Expression

Ning Ding , Chieri Tomomori-Sato§, Shigeo Sato§, Ronald C. Conaway§, Joan W. Conaway§, and Thomas G. Boyer 1

From the Institute of Biotechnology and Department of Molecular Medicine, The University of

Texas Health Science Center at San Antonio, San Antonio, Texas 78245, and the §Stowers

Institute for Medical Research, Kansas City, Missouri 64110 and the Department of

Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, Kansas 66160.

Running Head: MED19/MED26 are Synergistic Functional Targets of REST 1To whom correspondence should be addressed: 19715 Lambda Drive, San Antonio, Texas 78245. Tel: 210-567-7258; Fax: 210-567-7247; E-mail: boyer@uthscsa.edu.

A key hub for the orchestration of

epigenetic modifications necessary to restrict

neuronal gene expression to the nervous system

is the RE1 Silencing Transcription Factor

(REST; also known as Neuron Restrictive

Silencer Factor, NRSF). REST suppresses the

non-specific and premature expression of

neuronal genes in non-neuronal and neural

progenitor cells, respectively, via recruitment of

enzymatically diverse corepressors, including

G9a histone methyltransferase (HMTase) that

catalyzes di-methylation of histone 3-lysine 9

(H3K9me2). Recently, we identified the RNA

polymerase II transcriptional Mediator to be an

essential link between RE1-bound REST and

G9a in epigenetic suppression of neuronal genes

in non-neuronal cells. However, the means by

which REST recruits Mediator to facilitate

G9a-dependent extra-neuronal gene silencing

remains to be elucidated. Here, we identify the

MED19 and MED26 subunits in Mediator as

direct physical and synergistic functional

targets of REST. We show that although REST

independently binds to both MED19 and

MED26 in isolation, combined depletion of both

subunits is required to disrupt the association

of REST with Mediator. Furthermore,

combined, but not individual, depletion of

MED19/MED26 impairs REST-directed

recruitment to RE1 elements of Mediator and

G9a, leading to a reversal of G9a-dependent

H3K9me2 and de-repression of REST-target

gene expression. Together, these findings

identify MED19/MED26 as a probable

composite REST interface in Mediator and

further clarify the mechanistic basis by which

Mediator facilitates REST-imposed epigenetic

restrictions on neuronal gene expression.

The specification and maintenance of

neuronal identity within the developing vertebrate

nervous system derives from the influence of both

genetic and epigenetic programs that combine to

establish unique spatiotemporal patterns of

neuronal-specific gene expression. Expressed

genes that confer unique and highly specialized

morphological, biochemical, and physiological

properties on individual neuronal subtypes must be

suppressed in non-neuronal tissues, and the

regulatory mechanisms that coordinate these

processes are fundamentally important for proper

nervous system development and function (1-3).

A key factor in the orchestration of epigenetic

modifications that restrict the expression of

neuronal genes to the nervous system is the RE1

Silencing Transcription Factor (REST; also known

as Neuron Restrictive Silencer Factor, NRSF) (4,

5).

REST is a Kruppel-type zinc finger

transcription factor that binds to a 21-bp RE1

silencing element present in over 900 human

http://www.jbc.org/cgi/doi/10.1074/jbc.M806514200The latest version is at JBC Papers in Press. Published on December 2, 2008 as Manuscript M806514200

Copyright 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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genes, many of which encode proteins with

dedicated roles in neuronal determination, identity,

and function (4-10). REST occupies a central role

in non-neuronal lineage restriction through its

ability to suppress the non-specific and premature

expression of neuronal genes in non-neuronal cells

and neural progenitor cells, respectively (4, 5, 10,

11). Consistent with such a role, the expression of

REST is dominantly constrained in non-neuronal

and neural progenitor cells, although low levels of

REST protein are maintained in some populations

of postmitotic neurons, most notably those of the

hippocampus (10, 12-15). Functional inactivation

of REST in vertebrates leads to early embryonic

lethality and ectopic expression of neuronal genes

in non-neuronal tissues (16), whereas its forced

overexpression causes axon pathfinding errors

(17). Misregulation of REST-directed repression

has been linked with a variety of pathologic

conditions in humans, including Huntington’s

disease, epilepsy, ischemia, dilated

cardiomyopathy, X-linked mental retardation, and

cancer (18-30). Taken together, these observations

reveal fundamental links between REST and

vertebrate development and disease, and

emphasize the importance of a more

comprehensive understanding of REST-mediated

gene repression.

In this regard, REST has previously been

characterized as a bipartite transcriptional

repressor harboring two spatially and functionally

distinct repression domains: one spanning its N-

terminal 83 amino acids and a second

encompassing its C-terminal zinc finger (31-36).

Mechanistically, the N- and C-terminal repression

domains in REST have been shown to exert

repressive activity through recruitment of the

SIN3/HDAC and CoREST/HDAC/LSD1

corepressor complexes, respectively, both of

which function to impose restrictive epigenetic

modifications on the chromatin structure of REST-

target genes (31-36).

Recently, we identified a comparably

potent, yet previously uncharacterized, internal

repression domain in REST (amino acids 141-600)

encompassing its DNA-binding domain followed

by a lysine-rich region (21). We found that REST

(141-600) directly recruits a distinct corepressor

complex comprising Mediator, a multisubunit

global coregulator of RNA polymerase II

transcription, and G9a HMTase, an enzyme

dominantly responsible for transcriptionally

repressive histone-3 lysine-9 mono- (H3K9m) and

di-methylation (H3K9me2) within mammalian

euchromatin (21). In contrast to the well-

established role of Mediator as a bridge between

DNA-bound activators and the RNA polymerase II

general transcription machinery, our findings

revealed a critical requirement for Mediator in

recruitment of enzymatically active G9a by RE1-

bound REST, thus revealing Mediator to be a

direct link between REST and G9a-dependent

H3K9me2 required for extra-neuronal gene

silencing (21). Nonetheless, key elements of this

repressive protein interaction network remain to be

established, including the identity of the Mediator

subunit(s) with which REST directly interfaces to

recruit Mediator/G9a onto RE1 elements.

Here, using an unbiased in vitro protein

interaction screen to identify REST-binding

subunits in Mediator, we identified MED19 and

MED26 as candidate REST-target subunits. We

validated independent association of REST with

both MED19 and MED26 in isolation, but

nonetheless found that combined depletion of both

subunits was required to disrupt the association of

REST with Mediator. Furthermore, we found that

combined, but not individual, depletion of

MED19/MED26 impairs REST-directed

recruitment to RE1 elements of Mediator, G9a,

and G9a-dependent H3K9me2, leading to de-

repression of REST-target genes in vivo.

Collectively, these findings identify MED19 and

MED26 as synergistic physical and functional

targets of REST in Mediator and further clarify the

mechanistic basis by which Mediator facilitates

REST-imposed epigenetic restrictions on neuronal

gene expression.

EXPERIMENTAL PROCEDURES

Plasmids- Plasmids for in vitro

transcription/translation and/or mammalian

expression of REST, MED1, 6, 7, 8, 9, 10, 11, 12,

14, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29

and CDK8L have been described (37-45).

pCS3+CDK8-FLAG, pCS3+CycC-FLAG,

pCS3+MED31-FLAG, pCS3+MED16-FLAG and

pCS3+MED17-FLAG were constructed by

subcloning PCR-amplified corresponding cDNAs

into XhoI/ClaI linearized pCS3+ vectors bearing

FLAG epitope tag sequence. pCS2+MED4-His6-

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FLAG and pCS2+MED30-His6-FLAG were

constructed by subcloning PCR-amplified cDNAs

encoding MED4 and MED30, respectively, into

EcoRI/ClaI linearized pCS2+ vectors bearing

6XHis and FLAG epitope tag sequences.

pCS2+CBP-MED13 was constructed by

subcloning a PCR-amplified MED13 cDNA into a

pCS2+ vector bearing CBP (Calmodulin Binding

Peptide) epitope tag sequence. REST truncation

derivatives used in GST pull-down assays and

transient reporter-based transcriptional repression

assays have also been described (21).

Antibodies- Antibodies used for

immunoprecipitation and western blot analyses

correspond to the following: MED1 (sc-8998 &

sc-5334), MED6 (sc-9443), CDK8 (sc-1521),

REST (sc-15118), MED16 (sc-5366), MED17 (sc-

12453) and MED26 (sc-81237) were purchased

from Santa Cruz Biotechnology; MED12 (A300-

774A) was purchased from Bethyl Laboratories;

MED15 (H00051586-M02) was purchase from

Abnova; MED23 (551175) and CCNC (558903)

were purchased from BD pharmingen; CDK8

(RB-018) was purchased from Lab Vision Corp.;

G9a (G6919) antibody were purchased from

Sigma; H3K9me2 (07-441) were purchased from

Upstate. Murine HA monoclonal antibody was

purchased from Roche. Production and

purification of murine G9a and rabbit MED4/30

polyclonal antibodies has been described (21, 45).

Rabbit polyclonal anti-MED19 serum has also

been described (42, 44).

Cell Culture, Transfections, RNA

interference, and Reporter Assays- HeLa cells

were obtained from American Type Culture

Collection and cultured in DMEM (Invitrogen)

medium with 10% bovine growth serum (Hyclone).

DNA transfections were performed using Fugene

6 (Roche) and siRNA transfections using TransIT-

siQUEST (Mirus Bio Corp.) transfection reagents

following the manufacturer’s instructions. For

siRNA transfections, cells (~60% confluent) were

transfected with siRNAs at a final concentration of

20nM for 3 days before further analyses. siRNAs

(Dharmacon) correspond to the following: MED19

(J-016056-11); MED26 (J-011948-09); control

non-target siRNA (D-001210-01).

GST Pull-Down, Immunoprecipitation,

and Chromatin Immunoprecipitation Analyses -

For GST pull-down assays using radiolabeled

recombinant Mediator subunits or HeLa nuclear

lysates, GST derivatives were immobilized on

glutathione-Sepharose beads and washed

extensively with Lysis 250 buffer (50 mM Tris-

HCl, 250 mM NaCl, 5 mM EDTA) containing

0.5% Triton X-100 prior to incubation with either

radiolabeled Mediator subunits or HeLa nuclear

lysates [dialyzed against 0.1 M KCl D buffer (20

mM HEPES, pH 7.9, 0.2 mM EDTA, 20%

glycerol) for no less than 4 hours]. Beads were

washed 5 times with 0.3 M KCl D buffer

containing 0.2% NP-40 and eluted with Laemmli

sample buffer followed by SDS-PAGE and WB or

autoradiography analysis. For

immunoprecipitation of intact Mediator, nuclear

lysates (0.5mg) prepared as described previously

(45) were adjusted to 0.1 M KCl and 0.1% NP-40

and subjected to overnight immunoprecipitation at

4° C using protein A-Sepharose conjugated to

anti-MED4 antibody. Immunoprecipitates were

washed 5 times with 0.3 M KCl D buffer

containing 0.2% NP-40, eluted in Laemmli sample

buffer, and processed by SDS-PAGE for western

blot analysis. For chromatin immunoprecipitation

assays, cells were crosslinked and harvested into

cell lysis buffer (5mM HEPES pH 7.9, 85 mM

KCl and 0.5% Triton X-100) and then pelleted and

resuspended in nuclei lysis buffer (50mM Tris-

HCl pH 8.0, 10mM EDTA pH 8.0 and 1% SDS).

Chromatin was solubilized and sheared by pulsed

sonication (Fisher Scientific, Model 100) and

clarified by high-speed centrifugation. Chromatin-

containing fractions were diluted 10-fold in

dilution buffer (50mM Tris-HCl pH 8.0, 2mM

EDTA pH 8.0, 150mM NaCl and 1% Triton X-

100) followed by incubation with primary

antibodies as indicated at 4°C overnight. Immune

complexes were precipitated with a pre-blocked

mix of protein G-agarose and protein A-sepharose

for 2 hours followed by sequential washes with

sarcosyl buffer (TE and 0.2%Sarcosyl), low-salt

buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA

pH 8.0, 20 mM Tris-HCl pH 8.0, and 150 mM

NaCl), high-salt buffer (0.1% SDS, 1% Triton X-

100, 2 mM EDTA pH 8.0, 20mM Tris-HCl pH 8.0,

and 500 mM NaCl), LiCL detergent buffer (10

mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 1%

Deoxycholate, 1% NP-40, and 250 mM LiCl) and

TE. DNA was recovered in elution buffer (1%

SDS, 50 mM Tris-HCl pH 8.0 and 10 mM EDTA

pH 8.0) and subjected to proteinsae K treatment

and decrosslinking at 65°C overnight. DNA was

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purified by phenol/chloroform (1:1) extraction and

ethanol precipitation and resuspended in double

distilled water. RE1 occupancy levels are

expressed relative to RE1 occupancy levels in

control siRNA transfected cells. Primer sequences

for quantitative chromatin immunoprecipitation

assays have been described (21).

Reverse transcription-qPCR (RT-qPCR)

Analyses- RT-qPCR analyses have been described

previously (21). Briefly, RNA extracted from

HeLa cells transfected 3 days prior with specified

siRNAs, was subjected to reverse transcription and

real-time PCR analyses Results represent the

average of three independent experiments

performed in duplicate. mRNA levels are

expressed relative to mRNA levels in control

siRNA-transfected cells. Sequences of primers

used in RT-qPCR analyses are as follows: -Actin

(5’-CAAAGACCTGTACGCCAACACAGT-3’

and 5’-ACTCCTGCTTGCTGATCCACATCT-3’);

MED19 (5’-

TGGTTCCCATGATAACAGCAGCCT-3’ and

5’-CGGCTCTGTTTGTGCTTGTGCTTA-3’);

MED26 (5’-

AAACCTCTGACCCAGAAAGAGCCA-3’ and

5’- ACAGCTCCTTCCAGTTCGTCTGTT-3’);

SNAP25 (5’-AACTGGAACGCATTGAGGAAG-

3’ and 5’-GGTCCGTCAAATTCTTTTCTGC-3’);

Syn1 (5’-GGTCTCTGAAGCCGGATTTTG-3’

and 5’-GTCCCCAGTTTCTTATGCAGTC-3’);

M4 (5’-TTCATCCAGTTCCTGTCCAACCCA-3’

and 5’-GGCTTCTTGACGCTCTGCTTCATT-3’).

RESULTS

REST independently binds to MED19 and

MED26 both in vitro and in vivo - In an initial

attempt to identify the REST target subunit(s) in

Mediator, we screened 31 of 33 possible

mammalian Mediator subunits for in vitro

interaction with a GST-REST derivative

expressing REST amino acids 141-600 and

therefore encompassing the REST DNA-binding

domain followed by a lysine-rich region. Recently,

we showed that this domain harbors autonomous

repression activity in a manner requiring its direct

interaction with Mediator (21). Mediator subunits

omitted from this screen included MED12L and

MED13L, for which cDNAs are currently

unavailable. Among the remaining Mediator

subunits tested, only MED19 and MED26

exhibited significant REST binding activity, with

MED19 possessing an apparent greater affinity for

REST than MED26 (supplemental Figs. S1-S3).

Interestingly, comparative genomic analyses

indicate that whereas MED19 is broadly

represented throughout the eukaryotic kingdom,

with identifiable orthologs present in species

spanning humans to fungi, MED26 appears to be

restricted to higher eukaryotes, as no identifiable

ortholog is present in fungi.

To confirm the interaction specificities of

MED19 and MED26 for REST amino acids (aa)

141-600, we also tested these two Mediator

subunits for their respective abilities to bind to

REST N-terminal (aa 1-140) and C-terminal (aa

601-1098) fragments. Notably, MED19 and

MED26 bound only to REST (141-600)

corresponding precisely to the Mediator-binding

domain on REST (Fig. 1A) (21). To determine if

REST binds to MED19 and MED26 in vivo, we

examined the ability of REST to

coimmunoprecipitate along with either MED19 or

MED26 following their ectopic expression in HEK

293 human embryonic kidney cells. This analysis

revealed specific and efficient precipitation of

MYC-REST by FLAG-specific antibodies only in

the presence, but not in the absence, of either

FLAG-MED19 or FLAG-MED26 (Fig. 1B).

Taken together, the results of in vitro and in vivo

binding analyses reveal that REST (141-600)

interacts specifically and selectively with both

MED19 and MED26, suggesting that either one or

both of these subunits might represent a

functionally important target(s) of REST in

Mediator.

MED19 and MED26 synergistically

mediate the interaction between REST and

Mediator – As a precondition for exploring

potential functional interactions between REST

and MED19/MED26 through the use of RNAi in

mammalian cells, we evaluated the impact of

depleting either or both of these subunits on the

integrity of Mediator. To this end, we

immunoprecipitated Mediator from HeLa cells

following siRNA-mediated depletion of MED19

and/or MED26, and examined the

immunoprecipitates for the presence of Mediator

subunits representing each of four structurally

apparent Mediator subdomains (Head, Middle,

Tail, and Kinase) (46-48). We observed that

neither individual nor combined depletion of

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MED19/MED26 significantly altered the apparent

stability of other Mediator subunits examined or

their stable incorporation into Mediator (Fig. 2).

This finding is consistent with the prior

observation that Saccharomyces cerevisiaie

Mediator isolated under physiological conditions

from a MED19-deficient yeast strain is not

structurally compromised (49). Thus, under

physiological conditions, neither MED19 nor

MED26 are likely to be essential for the structural

integrity of Mediator.

Next, we examined the ability of Mediator

deficient in MED19 and/or MED26 to associate

with REST. To this end, GST-REST (141-600)

was tested for its ability to bind Mediator present

in nuclear extracts from HeLa cells depleted of

MED19 and/or MED26 by RNAi. Consistent with

our recent delineation of REST amino acids 141-

600 as Mediator-binding domain (21), we

confirmed that GST-REST (141-600) bound

Mediator present in nuclear extracts from HeLa

cells transfected with control siRNA (Fig. 3, lanes

1 and 5). Notably, we observed that only

combined, but not individual, depletion of

MED19/MED26 significantly impaired the

interaction between Mediator and REST (141-600)

(Fig. 3, lanes 2-4 and 6-8). These results confirm

that REST physically associates with Mediator,

likely through both its MED19 and MED26

subunits.

MED19 and MED26 are synergistically

required for REST repressor activity – Based on

our recent finding that REST-dependent

recruitment of Mediator is essential to link RE1-

bound REST with G9a in epigenetic gene

silencing, we hypothesized that the interaction

between REST (141-600) and MED19/MED26

might therefore be critical for REST-directed

repression. As a direct test of this hypothesis, we

investigated the impact of MED19 and/or MED26

depletion on the repressive activity of REST (141-

600) using a transient reporter-based

transcriptional repression assay. To this end,

REST (141-600), tethered to the GAL4 DNA-

binding domain, was tested for its ability to

repress transcription from a constitutively active

GAL4-responsive reporter plasmid in HeLa cells

depleted of MED19 and/or MED26 by RNAi. As

controls for these experiments, we also monitored

the impact of MED19 and/or MED26 depletion on

the respective repressive activities of the REST N-

and C-terminal repression domains, neither of

which binds to Mediator (21). Although individual

depletion of MED19 and MED26 had little impact

on the repressive activity of REST (141-600),

combined depletion of both Mediator subunits

dramatically impaired REST (141-600) repressor

activity (Fig. 4A, left panel). By contrast, neither

individual nor combined depletion of

MED19/MED26 significantly influenced the

repressive activities of the REST N- or C-terminal

repression domains (Fig. 4A, right panel). The

concordant MED19/MED26 requirement by REST

(141-600) for both Mediator binding and

Mediator-dependent transcriptional repression

supports the notion that MED19/MED26 are

physical and functional targets of REST in

Mediator.

To confirm the functional interaction

between REST and MED19/MED26 under more

physiological conditions, we monitored the impact

of MED19 and/or MED26 knockdown on the

expression levels of REST-repressed neuronal

genes in their natural chromosomal loci. In this

regard, we recently identified a Mediator

requirement for REST-directed G9a-dependent

repression of the SNAP25, Syn1, and M4 genes in

HeLa cells (21). Therefore, we used RT-qPCR

analyses to monitor the expression levels of these

three REST-target genes following RNAi-

mediated depletion of MED19 and/or MED26.

Consistent with findings from transient repression

assays, we observed that only combined, but not

individual, depletion of MED19/MED26 disrupted

REST repressor function, resulting in de-

repression of the SNAP25, Syn1, and M4 genes in

HeLa cells (Fig. 4B). Taken together, these

findings support the notion that MED19 and

MED26 are functionally important targets of

REST in Mediator and synergistically modulate

REST-directed repression in vivo.

MED19 and MED26 are synergistically

required for REST-directed recruitment of

Mediator, G9a, and H3K9me2 to RE1 elements

within REST-repressed neuronal genes – Recently,

we showed that the REST/Mediator/G9a

repressive network is conserved in a broad range

of non-neuronal cell types, and chromatin

immunoprecipitation (ChIP) analyses further

confirmed specific occupancy of RE1 elements

within REST-repressed neuronal genes by

REST/Mediator/G9a in the absence of markers of

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gene activation (21). Mechanistically, we showed

that Mediator recruited by RE1-bound REST

facilitates the deposition of transcriptionally

repressive H3K9me2 by G9a, with which

Mediator directly interacts through its MED12

interface (21). Therefore, we sought to investigate

whether MED19/MED26, as a probable REST

interface in Mediator, is required for REST-

directed recruitment of Mediator and G9a-

dependent H3K9me2 to RE1 silencing elements in

vivo. To address this question, we monitored the

transcription factor binding and histone

methylation profiles of the SNAP25, Syn1, and

M4 genes in HeLa cells as a function of MED19

and/or MED26 using RNAi and quantitative

chromatin immunoprecipitation (qChIP). This

analysis revealed that only combined, but not

individual, depletion of MED19/MED26

significantly impaired REST-directed recruitment

of Mediator, as well as G9a and G9a-dependent

H3K9me2 on all three REST-target genes (Fig. 5).

Taken together, our results reveal MED19/MED26

to be a crucial interface in Mediator necessary to

link RE1-bound REST with G9a in epigenetic

silencing of neuronal gene expression.

DISCUSSION It is now well established that Mediator is

a primary conduit of regulatory information

conveyed by gene-specific transcription factors to

RNA polymerase II. In this capacity, Mediator

plays an essential function in regulating the

assembly and/or activity of RNA polymerase II

transcription complexes on core promoters.

However, whether and how Mediator might

influence transcription factor-driven chromatin

modifications that impact RNA polymerase II

transcription has not been clear. In this regard, we

recently described a novel role for Mediator in

G9a-dependent epigenetic silencing of neuronal

gene expression imposed by the RE1 silencing

transcription factor REST. We showed that REST,

Mediator, and G9a form a trimeric complex in

vivo, that G9a binds to Mediator through its

MED12 interface, and that the MED12 interface in

Mediator is thus essential for REST-directed

recruitment of G9a and the imposition of

transcriptionally repressive H3K9me2 within

REST-targeted neuronal genes (21). Here, we have

extended these findings through the identification

MED19/MED26 as a direct interface for REST in

Mediator, thus providing a more complete

description of the physical and functional

interactions within the REST/Mediator/G9a

network required for extra-neuronal gene silencing.

First, we found that although REST binds to both

MED19 and MED26 in isolation, combined

depletion of both subunits is required to disrupt the

association of REST with Mediator. Second, we

found that combined, but not individual, depletion

of MED19/MED26 impairs REST-directed

recruitment of Mediator and G9a to RE1 elements,

leading to a reversal of G9a-dependent epigenetic

marks and de-repression of REST-target gene

expression. Because our results further reveal that

MED19 and MED26 do not depend upon one

another for incorporation into Mediator and their

concomitant loss does not disrupt the apparent

integrity of intact Mediator, these finding suggest

that MED19/MED26 comprise a probable

composite interface in Mediator for synergistic

physical and functional association with REST.

Combined with our previous findings (21), the

data presented here establish a model in which

Mediator, much like a molecular clamp, functions

to strengthen the interaction between REST and

G9a, thus facilitating the imposition of

transcriptionally repressive H3K9me2 around RE1

elements within REST-repressed neuronal genes.

In this model, REST and G9a interact with

Mediator through two distinct interfaces,

MED19/MED26 and MED12, respectively, and

both interactions are required for recruitment of

G9a to RE1-bound REST (Fig. 6).

To our knowledge, the identification

herein of MED19/MED26 as direct physical and

functional targets of REST represents the first

instance in which either Mediator subunit has been

shown to be a direct interface for gene-specific

transcription factors. In this regard, it is well

documented that enhancer-bound activators can

recruit Mediator through individual subunits to

facilitate the assembly and/or stimulation of

transcription preinitiation complexes on core

promoters (50, 51). For example, members of the

nuclear receptor superfamily recruit Mediator via

direct interactions with discrete LxxLL motifs

within its MED1 subunit (52-56), while another

class of activators, including adenovirus E1A, Esx,

Elk-1 and C/EBP instead target Mediator through

its MED23 subunit (57-60). Furthermore, MEDs

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12, 14, 15, 16, 17, and 25 have each been

implicated as direct physical and functional targets

within Mediator of various gene-specific

transcriptional activators (38, 45, 61-71). Our

discovery that Mediator, through its

MED19/MED26 interface, is recruited by RE1-

bound REST to silence neuronal genes thus

provides a repressive corollary to the well-

characterized mechanism of activator-dependent

Mediator recruitment.

Recently, a comprehensive comparative

genomics analysis of Mediator across the

eukaryotic kingdom revealed that MED19 and

MED26, along with other MED subunits, arose

early on during eukaryotic diversification (72). It

has been proposed that the evolutionary addition

of these “peripheral” subunits onto an existing

core 17-subunit Mediator proto-complex present

1-2 billion years ago could have accommodated

unique transcriptional mechanisms that underlie

the advanced genetic circuitry of multicellular

organisms (72). In this regard, it is perhaps notable

that whereas MED19 is broadly represented

throughout eukaryotes, with identifiable orthologs

present in metazoa, fungi, and plants, amoebae,

and diatoms, MED26 is restricted to metazoans

and amoebae (72). Within the metazoa, MED26 in

fact exhibits a species distribution that closely

overlaps that of REST, possibly suggesting that

the interaction of REST with a MED19/26

Mediator interface arose as a consequence of co-

evolution and contributed to the diversification

and development of metazoans.

An unexpected finding to emerge from

this study was the identification of MED26 as a

physical and functional target of REST. This

observation implicates MED26 directly in

transcriptional repression and thus challenges a

common conception that MED26 functions

exclusively in transcriptional activation. A

principal activating role for MED26 in

transcription control has been invoked largely on

the basis of its observed preferential association

with an active form of Mediator biochemically

isolated from mammalian cells. Thus, elegant

structural and functional analyses have previously

revealed that a MED26-proficient Mediator

species devoid its kinase module is capable of

supporting activator-dependent RNA polymerase

II transcription in vitro, whereas a MED26-

deficient Mediator species that additionally

includes the kinase module inhibits this process

(57, 73, 74). Nonetheless, how these two

biochemcially stable isolates relate to the possible

range of dynamic Mediator complexes assembled

on target gene promoters in vivo remains to be

definitively established. It is possible, for example,

that the structurally and functionally distinct

Mediator species isolated biochemically represent

static extremes across a continuum of dynamic

Mediator complexes assembled in vivo. This

possibility is supported by the results of recent

proteomics analyses indicating that the association

of MED26 and the kinase module with core

Mediator is not, in fact, mutually exclusive (74,

75). This observation suggests the existence of an

intermediate or “transition” state of Mediator, one

in which incorporation of all possible subunits is

accommodated. We speculate that the Mediator

complex recruited by REST could reflect this

intermediate state, thus providing REST with a

Mediator platform of sufficient functional

complexity to exert complex regulation of

neuronal gene expression. In this regard, our

identification of MED26 as a direct target of

REST might help to explain the comparably

poorly understood role of REST in context-

dependent transcriptional activation (12, 16, 76-

78).

Our studies revealed that combined

depletion of MED19/MED26 impaired REST-

directed, G9a-dependent imposition of

transcriptionally repressive H3K9me2 to an extent

that far exceeded the additive impact of

individually depleting either subunit alone,

suggesting that MED19/MED26 function

synergistically to mediate REST-directed neuronal

gene silencing. Previous studies have documented

the ability of gene-specific transcriptional

activators to target more than one subunit in

Mediator. For example, in mammals, the

glucocorticoid receptor binds to MED1 and

MED14 (79), the retinoid X receptor and the

retinoic acid receptor both bind to MED1 and

MED25 (71), p53 binds to MED1 and MED17

(80, 81), and VP16 binds to MED17 and MED25

(38, 70, 80). In plants, LEUNIG has been shown

to bind to MED14 and CDK8 (82). However, in

none of these instances has functional synergy

between different Mediator subunits targeted by

the same activator been demonstrated. Thus, the

identification herein of MED19 and MED26 as

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dual functional targets of REST represents the first

example of functional synergy among Mediator

subunits targeted by a common transcriptional

regulator. Why does REST require physical

interaction with both MED19 and MED26 in

Mediator? One possibility is that REST is such an

important developmental regulator that sufficient

mechanistic redundancy within its regulatory

networks must exist for REST to efficiently

perform its function throughout the genome.

Further studies will be required to elucidate the

topological basis for functional synergy within

Mediator and further clarify the fundamental logic

that drives REST-dependent developmental gene

regulation.

In summary, our findings strongly suggest

that REST recruits Mediator through a

MED19/MED26 interface in order to facilitate

epigenetic silencing of neuronal genes in non-

neuronal cells. This work thus identifies MED19

and MED26 as critical components of the

regulatory apparatus employed by REST to restrict

neuronal gene expression to the nervous system

and thereby contribute to the specification of

neuronal identity.

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FOOTNOTES

* This work was supported in part by Public Health Service grant CA-0908301 from the National Cancer

Institute (T.G.B).

FIGURE LEGENDS

FIGURE 1. MED19 and MED26 bind specifically to REST. A, Recombinant full-length MED19 and

MED26 proteins were expressed and radiolabeled with [35

S]methionine by translation in vitro prior to

incubation with glutathione-Sepharose-immobilized GST or GST-REST derivatives as indicated. Bound

proteins were eluted with Laemmli sample buffer, resolved by 15% SDS-PAGE, and visualized by

phosphorimager analysis. Input, 10% of each in vitro translated protein used in binding reactions. The

amount of MED19 and MED26 bound by GST-REST (141-600) was quantified and expressed as a

percentage of the total input. Results are representative of at least three independent binding experiments.

B, Myc-REST was expressed with or without FLAG-MED19 or FLAG-MED26 in HEK 293 cells prior to

immunoprecipitation (IP) of whole cell lysates using antibodies specific for the FLAG epitope as

indicated. Immunoprecipitates were resolved by SDS-PAGE and processed by western blot (WB)

analysis using FLAG- or Myc-specific antibodies as indicated. Input, 10% of the nuclear lysate used for

IP reactions. Asterisk indicates the position of IgG heavy chain.

FIGURE 2. MED19 and MED26 are not essential for the apparent integrity of Mediator. Nuclear

lysates from HeLa cells transfected with control (CNTL), MED19-, MED26-, or MED19&26-specific

siRNAs were subjected to immunoprecipitation (IP) using antibodies specific for MED4.

Immunoprecipitates were extensively washed prior to resolution by SDS-10% PAGE and processing by

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western blot (WB) analysis using the specified antibodies. Input, 10% of the nuclear lysates used for IP

reactions. Structural domains to which individual Mediator subunits may be relegated are indicated [Head,

Middle, Tail, Kinase, or Unassigned (Unass.)]. Asterisks mark MED19 and MED26 Mediator subunits

targeted for RNAi-mediated depletion.

FIGURE 3. MED19 and MED26 synergistically mediate the interaction between REST and

Mediator. Nuclear lysates from HeLa cells transfected with control (CNTL), MED19-, MED26-, or

MED19&26-specific siRNAs as indicated were incubated with immobilized GST-REST (141-600).

Specifically bound proteins were resolved by SDS- 10% PAGE prior to WB analysis using the specified

antibodies. Input, 10% of the nuclear lysates used in binding reactions.

FIGURE 4. MED19 and MED26 are synergistically required for REST-directed transcriptional

repression. A, HeLa cells were transfected with control, MED19-, MED26-, or MED19&26-specific

siRNAs as indicated 48 hrs prior to co-transfection with pG5TK-Luc along with Gal4 or Gal4-REST

derivatives as indicated and subsequent assay of transfected whole cell lysates for normalized luciferase

activities. Luciferase activities are expressed relative to the luciferase activity obtained in cells transfected

with control siRNA and Gal4. Data represent the mean +/- SEM of at least three independent

transfections performed in duplicate. Asterisks denote statistically significant values relative to control

siRNA (Student’s t test, **P < 0.05). Gal4-REST-N and -C terminal derivatives express REST amino

acids 1-140 and 999-1098, respectively. Immunoblot analysis of transfected whole cell lysates revealed

that Gal4-REST derivatives were expressed at roughly equivalent levels (Supplemental Fig. S4). B, RNA

from HeLa cells transfected with control, MED19-, MED26-, or MED19&26-specific siRNAs as

indicated was used for RT-qPCR. mRNA levels are expressed relative to mRNA levels in control siRNA-

transfected cells, which was arbitrarily assigned a value of 1. Data represent the mean +/- SEM of at least

three independent experiments performed in duplicate. Asterisks denote statistically significant values

relative to control siRNA (Student’s t test, **P < 0.05).

FIGURE 5. MED19 and MED26 are synergistically required for recruitment of Mediator and G9a-

dependent H3K9me2 by RE1-bound REST. Soluble chromatin prepared from HeLa cells transfected

with control, MED19-, MED26-, or MED19&26-specific siRNAs as indicated was subjected to IP using

the indicated antibodies. Immunoprecipitated chromatin was analyzed by quantitative PCR using primers

flanking RE1 elements within the M4, SNAP25, and Synapsin1 (Syn1) genes. The level of RE1 site

occupancy for each protein is expressed relative to its level of occupancy in control siRNA-transfected

cells, which was arbitrarily assigned a value of 100%. Data represent the mean +/- SEM of at least three

independent experiments performed in triplicate. Asterisks denote statistically significant values relative

to control siRNA (Student’s t test, **P < 0.05, ***P < 0.01).

FIGURE 6. Schematic model for the network of functional interactions among REST, Mediator and

G9a required for epigenetic silencing of neuronal gene expression. RE1-bound REST recruits

Mediator through its MED19/MED26 subunits. Mediator, in turn, facilitates recruitment of G9a-

dependent H3K9me2 through direct interaction of G9a with the MED12 interface in Mediator. Although

REST can bind directly to G9a in vitro, it nonetheless requires Mediator for recruitment of G9a and the

imposition of transcriptionally repressive H3K9me2 in vivo (21).

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MED19

Inpu

tG

ST

1-14

014

1-60

060

1-10

98

REST

% Bound

25.1

MED26 3.2

Coomassie

GST

1-140

141-600

601-1098

Figure 1

A B

WB:FLAG

WB:Myc

*

Myc-RESTFLAG-MED19FLAG-MED26

+ + + + + ++ +

+ +-- -

--- -

-

10% Input FLAG-IP

F-MED26

F-MED19

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MED23

MED12CDK8CCNC

MED1MED4

MED6

MED16

MED17

MED26*

MED15

MED19*

Figure 2

siC

NTL

siM

ED

19si

ME

D26

siM

ED

19/2

6

siC

NTL

siM

ED

19si

ME

D26

siM

ED

19/2

6

Input MED4 IP

WB:

Kin

ase

Hea

dM

iddl

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ilU

nass

.

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siC

NTL

siM

ED

19si

ME

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siM

ED

19/2

6

REST-(141-600)

Figure 3

siC

NTL

siM

ED

19si

ME

D26

siM

ED

19/2

6

Input

MED12MED23MED1CDK8

MED26MED19

WB:

1 2 3 4 5 6 7 8

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Gal4 REST(141-600)

RLU

00.20.40.60.81.01.21.4

REST-N REST-CGal400.20.40.60.81.01.21.41.6

RLU

β-Actin MED19 MED26 SNAP25 Syn1 M4

Rel

ativ

em

RN

ALe

vel

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Figure 4A

B

siRNA: Control MED19 MED26 MED19/26

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siRNA: Control MED19 MED26 MED19/26

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REST G9a H3K9me2 MED4 MED30 MED120

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Figure 5

REST G9a H3K9me2 MED4 MED30 MED12

REST G9a H3K9me2MED4 MED30 MED12

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siRNA: Control MED19 MED26 MED19/26 by guest on M

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Figure 6

Mediator

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H3K9me2RE1 element

Neuronal Genes OFF

REST

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and Thomas G. BoyerNing Ding, Chieri Tomomori-Sato, Shigeo Sato, Ronald C. Conaway, Joan W. Conaway

transcription factor in epigenetic silencing of neuronal gene expressionMED19 and MED26 are synergistic functional targets of the RE1 silencing

published online December 2, 2008J. Biol. Chem. 

  10.1074/jbc.M806514200Access the most updated version of this article at doi:

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Supplemental material:

  http://www.jbc.org/content/suppl/2008/12/09/M806514200.DC1

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